A rapid method for reconstitution of bacterial membrane proteins.
We have devised a simple method for the reconstitution of bacterial membrane proteins directly from small (1-20 ml) volumes of cell culture, thus eliminating the preparation of membrane vesicles. Cells are subjected to simultaneous lysozyme digestion and osmotic lysis, and after brief centrifugation ghosts are solubilized in 1.2% octyl-beta-D-glucopyranoside (octylglucoside) in the presence of added carrier lipid and an osmolyte. Aliquots of the clarified supernatant are suitable for reconstitution, as documented by using extracts from three different Gram-negative cells to recover both inorganic phosphate (Pi)-linked antiport and oxalate:formate exchange activities in proteoliposomes. These proteoliposomes are physically stable, non-leaky and can sustain a membrane potential and, because functional porins do not reconstitute, the artificial system has transport characteristics similar to those found when proteoliposomes are obtained using standard methods. This method should become an important tool for the screening and characterization of large numbers of strains, both wild-type and mutant.
Varadhachary
A
A
Department of Physiology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Maloney
P C
PC
D001426:Bacterial Proteins / Q000378:metabolism; D001433:Bacteriolysis; D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry; D002498:Centrifugation; D006090:Gram-Negative Bacteria / Q000032:analysis* / Q000648:ultrastructure; D007474:Ion Exchange; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism; D008722:Methods; D009113:Muramidase / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism
D001426:Bacterial Proteins; D008565:Membrane Proteins; D010710:Phosphates; D011510:Proteolipids; C020485:proteoliposomes; D009113:Muramidase